1364
J . Med. Chem. 1990,33, 1364-1368
Antitumor Agents. 11 1.7 New 4-Hydroxylated and 4-Halogenated Anilino Derivatives of 4’-Demethylepipodophyllotoxin as Potent Inhibitors of Human DNA Topoisomerase I1 Kuo-Hsiung Lee,*,$ Scott A. Beers,$ Masami Mori,: Zhe-Qing Wang,: Yao-Haur Kuo,* Li Li, Su-Ying Liu,s Jang-Yang Chang,§ Fu-Sheng Han,§ and Yung-Chi Chengs Natural Products Laboratory, Division of Medicinal Chemistry and Natural Products, School of Pharmacy, and Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599. Received July 5, 1989
A series of C-4 hydroxylated and halogenated anilino derivatives of epipodophyllotoxin or 4’-demethylepipodophyllotoxin have been synthesized and evaluated for their inhibitory activity against the human DNA topoisomerase I1 as well as for their activity in causing cellular protein-linked DNA breakage. Compounds 11-17 and 22 are more potent than etoposide in causing DNA breakage, while compounds 11-13, 15, 16, and 20 are as active or more active than etoposide in their inhibition of the human DNA topoisomerase 11. The cytotoxicity in KB cells appears to have no direct correlation with their ability to inhibit DNA topoisomerase I1 and to cause protein-linked DNA breaks in cells.
Etoposide (1) reached clinical study in the early 1970s. Its clinical efficacy has stimulated a renewed interest in the medicinal chemistry of podophyllotoxin-derived antitumor agents.2-8 It has recently been shown that 1 and related compounds are potent inhibitors of DNA topoisomerase 11. These drugs block the catalytic activity of DNA topoisomerase I1 by stabilizing a cleavable enzymeDNA complex in which the DNA is cleaved and covalently linked to the enzyme.*” Structure-activity relationship studies have demonstrated a direct correlation between cytotoxicity, DNA breakage, and mice-derived topoisomerase I1 inhibition activities among the podophyllotoxin analogues.12 In our previous study13 we found that substitution of the glycosidic moiety of 1 by an 4-alkylamino side chain, as in 4’-demethyl-4P-[ (2”-hydroxyethyl)amino]-4-desoxypodophyllotoxin,showed potent inhibitory activity on human DNA topoisomerase I1 as well as stronger activity in causing cellular protein-linked DNA breakage. The enzyme-inhibitory activity correlates reasonably well with its activity in causing protein-linked DNA breakage in KB cells. The in vitro cytotoxicity (KB) appears to have no correlation with the inhibitory activity of the human DNA topoisomerase 11. As part of our continuing efforts along this line, we describe herein the preparation and structure-activity relationships of a series of 4-arylamino compounds derived from 4’-demethylepipodophyllotoxin or podophyllotoxin. Chemistry The 4-arylamino derivatives of epipodophyllotoxin (8-10) were synthesized from podophyllotoxin (21, while the 4-arylamino derivatives of 4’-demethylepipodophyllotoxin (11-23) were prepared from 4’-demethylepipodophyllotoxin (3) (Scheme I). These syntheses employed a modification of Kuhn’s method.14 The key intermedi(4) and 4’-deates, 4~-bromo-4-desoxypodophyllotoxin methyl-4/3-bromo-4-desoxypodophyllotoxin (6), were ob-
tained via bromination of 2 and 3, respectively. They were (5) accompanied by 4/3-methoxy-4-desoxypodophyllotoxin and 4’-demethyl-4~-methoxy-4-desoxypodophyllotoxin (7). The formation of 5 and 7 were due to the replacement of the bromo atom in 4 and 6 with trace amounts of methanol present in dichloromethane. As the bromo intermediates (4 and 6) were highly reactive and susceptible toward ‘For part 110, see ref 1. Natural Products Laboratory, Division of Medicinal Chemistry and Natural Products. 8 Department of Pharmacology. Present address: Department of Pharmacology, School of Medicine, Yale University, New Haven. CT 06510.
*
0022-2623/90/1833-1364$02.50/0
nucleophilic attack even by moisture, and 5 and 7 were relatively stable toward the next step of the reaction, the crude bromination products (4 and 6) were subjected to the next step of the reaction to yield 8-23 without further purification. Thus, the compounds 8-23 were synthesized by nucleophilic substitution from the bromination products (4 and 6) with appropriate arylamines. Presumably, this substitution underwent via a SN1mechanism, which occurred on the C-4 benzylic carbonium ion. The bulky C-la pentant aromatic ring directed the substitution to be stereoselective, resulting in the formation of C-4O-oriented 8-23 as the main products, accompanied by C-4a isomers of 8-23. The ratio for the P- and a-isomers varied from a range of 6:l to 12:l.15 The assignment of the configuration at C-4 for 8-23 was based not only on the difference between the J3,4 coupling (1) Part 110: Yamagishi, T.; Haruna, M.; Yan, X. Z.; Chang, J. J.; Lee, K. H. J . Nat. Prod. 1989,52, 1071. (2) Jardine, I. In Anticancer agents based on natural product
models; Cassady, J. M., Douros, J., Eds.; Academic Press: New York, 1980; p 319 and references therein. (3) Issell, B. F.; Muggia, F. M.; Carter, S. K. Etoposide [ VP-161 current status and new developments. Academic Press: Orlando, 1984; pp 1-355. (4) Saito, H.; Yoshikawa, H.; Nishimura, Y.; Kondo, S.; Takeuchi, T.; Umezawa, H. Chem. Pharm. Bull. 1986,34, 3733. (5) Saito, H.; Nishimura, Y.; Kondo, S.; Komuro, K.; Takeuchi, T. Bull. Chem. SOC.Jpn. 1988,61, 2493. (6) Thurston, L. S.; hie, H.; Tani, S.; Han, F. S.; Liu, Z. C.; Cheng, Y. C.; Lee, K. H. J . Med. Chem. 1986,29, 1547. ( 7 ) Beers, S. A.; Imakura, Y.; Dai, H. J.; Li, D. H.; Cheng, Y. C.; Lee, K. H. J . Nat. Prod. 1988, 51, 901. (8) Thurston, L. S.; Imakura, Y.; Haruna, M.; Li, D. H.; Liu, Z. C.; Liu, S. Y.; Cheng, Y. C.; Lee, K. H. J. Med. Chem. 1989, 32, 604. (9) Chen, G. L.; Yang, L.; Rowe, T. C.; Halligan, B. D.; Tewey, K.; Liu, L. J . Biol. Chem. 1984, 259, 13560. (10) Ross, W.; Rowe, T.; Glisson, B.; Yalowich, J.; Liu, L. Cancer Res. 1984, 44, 5857. (11) Rowe, T.; Kuppfer, G.; Ross, W. Biochem. Pharmacol. 1985, 34, 2483. (12) Minocha, A.; Long, B. Biochem. Biophys. Res. Commun. 1984, 122, 165. (13) Lee, K. H.; Imakura, Y.; Haruna, M.; Beers, S. A.; Thurston, L. S.;Dai, H. J.; Chen, C. H.; Liu, S. Y.; Cheng, Y. C. J . Nat. Prod. 1989, 52, 606. (14) Kuhn, M.; Keller-Juslen, C.; Von Wartburg, A. Helu. Chim. Acta 1969, 52, 944. (15) When the reaction mixtures were fractionated on a silica gel column for purification, the C-40-substituted compounds were alwavs collected before C-40-substitution. 0 1990 American Chemical Society
Journal of Medicinal Chemistry, 1990, Vol. 33, No. 5 1365
Antitumor Agents Scheme I 9H
( : m ( o
H,CO
Q0 OCH, OCH,
NHAr
HBr
(.&o( & (:( ArNH,
H3C0Q OCH, o - OCH,
2
4, R = B r
H,CO
Q0 OCH, OCH3
8
- 10
5, R=OCH3
OH
OH
3
OH
6, R = B r 7, R = OCH,
constants13 but also on the different chemical shift for the proton at C-5. The C-4P-substituted products showed H-5 as a singlet a t 6 6.70-6.81, whereas, in the case of C-4asubstituted isomers, it appeared as a singlet at 6 7.00-7.20. This difference was clearly due to the anisotropic effect of the aromatic ring attached a t either the or the a C-4 amino group on H-5.
Results and Discussion Previous studies on the structure-activity relationships among podophyllotoxin and related compounds have suggested the following structural requirements for antitumor a c t i ~ i t y : (a) ~ , ~the presence of a 4’-phenolic hydroxyl group, (b) the presence of a glucoside moiety on the C-4 hydroxyl group and a 4”,6”-O-cyclicacetal moiety on the hexapyranose ring, (c) the j3-configuration at C-4 position, and (d) the trans-fused lactone ring. As illustrated in Table I, compounds 8-10 lacking a free OH group a t C-4’ showed no DNA scission activity and no inhibition on DNA topoisomerase 11. On the contrary, the 4’-demethyl derivatives (11-13) demonstrated potent activity in both models. This coincides very well with the above-mentioned structural requirement (a). The cytotoxicity of 8-10 could be due to their potential effects on tubulin polymerization as seen in podophyllotoxin. The lack of the correlation between cytotoxicity and induced protein-linked DNA breaks in cells by these compounds is not surprising. It was previously shown that etoposide-induced DNA break is insufficient to cause cell death.16 The possible existence of other biochemical determinants of those compounds’ action, such as their potential effects on tubulin polymerization in cells, could also contribute to the lack of correlation between these parameters. Furthermore, the cytotoxic action of the compound will also depend on the uptake of the compound into cells. (16) Long, B. H.; Musial, S. T.;Brattain, M. G. Biochemistry 1984. 23, 1183.
(17) Liu, S. Y.; Hwang,B. D.; Haruna, M.; Imakura, Y.; Lee, K. H.; Cheng, Y. C. Mol. Pharmacol. 1989, 36, 78.
11
- 23
The compounds (1 1-17,20,and 22) possessing no glucoside moiety a t the C-4 hydroxy group showed comparable or superior activity to 1. It clearly indicated that the replacement of the 4-0-glycosyl moiety by a simpler arylamino group still retained the activity of l. The glucosidic ethylidene cyclic acetal moiety was not so essential for the biological activity as mentioned above in (b). As described in our previous studies dealing with 4’demethyl-4~-(alkylamino)-4-desoxypodophyllotoxin~,~~ those compounds having a 0-hydroxyethylene chain (e.g. 4’-demethyl-4P-[ (2’’-hydroxyethyl)amino]-4-desoxypodophyllotoxin) showed potent inhibition on enzyme as well as on DNA strand breakage. Prolongation of the ethylene side chain, as in the hydroxypropyl analogue, led to reduction in potency. A similar situation was seen in compounds 11-13. The most active substance among them in the three assay systems (Table I) was 12. Compound 12 possessed an oxygen atom meta to the nitrogen atom. Although there are three carbon atoms separating the oxygen atom from the nitrogen atom, the bond distance in a benzene ring is shorter than that between sp3 hybridized carbon atoms. In addition, the bond distance between oxygen and sp2 carbons is less than that between sp3 hybridized carbon atoms. The same applies for the bond distance between nitrogen and sp2 vs nitrogen and sp3carbon atoms. The result is that the distance between oxygen and nitrogen in 12 is not as far as it was in 4’-demethyl-4P- [ (2/’-hydroxyethyl) amino] -4-desoxypodophyllotoxin. Table I also illustrated two factors necessary for optimum activity in three bioassay systems of the halogenated compounds (14-23) which are different from those of the hydroxyl compounds (11-13). First, the distance between the halogen and the nitrogen is important. The compound showed the greatest activity when the halogen (F, C1, Br, I) is in the para position. The activity then decreased as the halogen was moved into the meta position with the least activity occurring when the halogen is in the ortho position. The second factor involves the size of the substituted halogen. Although the distance-dependent activity relationship is still seen when fluorine (similar in size to
1366 Journal of Medicinal Chemistry, 1990, Vol. 33, No. 5
Lee et al.
Table I. Biological Evaluation of 4-(Arylamino)epipodophyllotoxins
no.
R2
cytotoxicity: IDw KB, 1M
inhibn of DNA topoisomerase I1 activity 100 pMb ID,: PM
cellular protein-DNA complex formation, 70 (10pM)
H
0.20
+++
8
CH3
4.11
+
6
9
CH3
0.31
+
37
10
CH3
0.59
+
21
11
H
4.54
++++
25
151
12
H
0.45
++++
5
290
13
H
2.26
+++
25
211
H
0.25
++
H
0.23
+++
50
158
H
0.24
++++
10
213
H
1.08
++
50
115
H
2.34
+
32
19
H
2.29
++
51
20
H
0.22
+++
21
H
2.36
+
22
H
0.22
++
23
H
0.34
+
1
OH
14
15
.;HN
I
'
F
F
F
-Q
HN I
F
I
121
-
18 HN
100
-
16 17
50
-
50
99 62
100
179 64
a ID, was the concentration of drug that affords 50% reduction in cell number after a 3-day incubation. * +, ++, +++, ++++, and denote 25%, So%, 75%, >%%, and 0% inhibition. CEachcompound was examined with three concentrations at 25,50, and 100 wM. The IDw value was established on the basis of the degree of inhibition at these three concentrations.
Antitumor Agents
hydrogen) is used as the halogen, all four fluorine compounds endow with comparable activity with 1 in three bioassay systems. Experimental Section General Experimental Procedures. All melting points were taken on a Fischer-Johns melting point apparatus and were uncorrected. IR spectra were recorded on a Perkin-Elmer 1320 spectrophotometer, and ‘H NMR spectra were obtained by using either a JEOL FX-60 or a Bruker 250 NMR spectrometer; all chemical shifts were reported in parts per million from TMS. Elemental analyses were performed by Galbraith Laboratories, Inc., Knoxville, TN, or by Atlantic MicroLab Inc., Norcross, GA. Mass spectral analyses were determined on a V.G. Micromass 70-70 instrument at 70 eV with a direct inlet system. Analytical thin-layer chromatography (TLC) was carried out on Merck precoated silica gel 60 F-254. EM Kieselgel 60 (230-400 mesh ASTM) was used for CC. Preparative TLC was performed on Analtech precoated silica gel GF (500 Fm, 20 X 20 cm). All new target compounds were characterized by melting point and ‘H NMR and IR spectral analyses as well as elemental analyses. Synthesis of Compounds 8-10. Podophyllotoxin (500 mg, 1.2 mmol) was dissolved in dry dichloromethane (10 mL) and kept at 0 “C. Hydrogen bromide gas was introduced into the solution for 45 min, after which time the solvent was evaporated in vacuo, and anhydrous tetrahydrofuran (15 mL), anhydrous barium carbonate (474 mg, 2.4 mmol), and the appropriate hydroxyanilines (142 mg, 1.3 mmol) were added. The mixture stood a t room temperature overnight and then was filtered and concentrated. The crude product was purified by column chromatography (silica gel (45 g) with dichloromethane-acetonethyl acetate 100:5:5 as an eluant). 4~-(2”-Hydroxyanilino)-4-desoxypodophyllotoxin(8): amorphous crystals from ether, mp 145-148 “C; IR (KBr) 3480 (OH), 3410 (NH), 2900 (aliphatic CH), 1760 (lactone), 1580,1475 (aromatic C=C) cm-’; ‘H NMR (CDC13)6 6.90 (t, J = 6.6 Hz, 1 H, 4”-H), 6.78 (s, 1 H, 5-H), 6.65 (m, 2 H, 3”,6”-H), 6.53 (m, 2 H, 8-H, 5”-H), 6.34 (s, 2 H, 2’,6’-H), 5.96 (AB q, J 1.0, 3.5 Hz, 2 H, OCHzO), 5.02 (s, 1 H, exchangeable, 2”-OH), 4.68 (m, 1 H, 4-H), 4.62 (d, J = 4.9 Hz, 1 H, 1-H), 4.38 (t, J = 8.6 Hz, 1 H, 11-H), 4.33 (m, 1 H, exchangeable, NH), 4.00 (t, J = 8.6 Hz, 1 H, 11-H), 3.82 (s,3 H, 4’-OCH,), 3.76 (s,6 H, 3’,5’-OCH,), 3.25 (dd, J = 5.1, 14.0 Hz, 1 H, 2-H), 3.05 (m, 1 H, 3-H); MS m/z = 505 (M’). Anal. (Cz8Hz7N08~3/zHz0) C, H. 4/3-(3-Hydroxyanilino)-4-desoxypodophyllotoxin(9): amorphous powder from ether, mp 148-150 OC; IR (KBr) 3370 (OH, NH), 2900 (aliphatic CHI, 1760 (lactone), 1585, 1475 (aromatic C=C) cm-’; ‘H NMR (CDClJ 6 7.05 (t, J = 8.0 Hz, 1 H, 5”-H), 6.77 (s, 1 H, 5-H), 6.52 (s, 1 H, 8-H), 6.32 (s,2 H, 2’,6’-H), 6.25 (dd, J = 2.2, 8.0 Hz, 1 H, 4”-H), 6.14 (dd, J = 2.2, 8.0 Hz, 1 H, 6”-H), 6.05 (t, J = 2.2 Hz, 1 H, 2”-H), 5.96 (AB q, J = 1.3, 3.8 Hz, 2 H, OCHZO), 4.64 (d, J = 3.9 Hz, 1 H, 4-H), 4.49 (d, J = 5.0 Hz, 1 H, 1-H), 4.4 (t, J = 8.7 Hz, 1 H, 11-H), 4.03 (t, J = 8.7 Hz, 1H, 11-H),3.81 (s,3 H, 4’-OCH3), 3.76 ( ~ , H, 6 3’,5’-OCH3), 3.18 (dd, J = 5.0 Hz, 14.0 Hz, 1 H, 2-H), 3.02 (m, 1 H, 3-H); MS . HH.~ O ) m/z = 505 (M’). Anal ( C Z ~ H ~ ~ N O ~C, 4 8 44”-Hydroxyanilino)-4-desoxypodophyllotoxin ( 10): crystals from chloroform, mp 145-150 “C; IR (KBr) 3310 (OH, NH), 3010 (aromatic CH), 2900 (aliphatic CHI, 1730 (lactone), 1575,1475(aromatic C-H) cm-’; ‘H NMR (CDCl,, DzOexchange) 6 6.75 (d, J = 8.3 Hz, 3 H, 5-H, 3”,5”-H), 6.53 (s, 1 H, 8-H), 6.45 (d, J = 8.3 Hz, 2 H, 2”,6”-H), 6.23 (s, 2 H, 2’,6’-H), 5.95 (AB q,
Journal of Medicinal Chemistry, 1990, Vol. 33, No. 5 1367
demethyl-4~-bromo-4-deoxypodophyllotoxin (6) (300 mg, 0.65 mmol), anhydrous barium carbonate (153 mg, 0.78 mmol), and the appropriate arylamine (0.78 mmol) in 7 mL of dry 1,2-dichloroethane under nitrogen was stirred overnight at room temperature. The reaction mixture was filtered, diluted with ethyl acetate, washed with water, dried, and purified via column chromatography (30 g of silica gel with dichloromethane-acetone-ethyl acetate 10055 or toluene-ethyl acetate 3:l as eluant). 4’-Demethyl-4~-(2”-hydroxyanilino)-4-desoxypodophyllotoxin (11): yield 28%; crystals from ether, mp 175 “C; IR (KBr) 3360 (OH, NH), 2900 (aliphatic C-H), 1750 (lactone), 1600, 1475 (aromatic C=C) cm-’; ‘H NMR (CDCl,) 6 6.88 (t, J = 7.4 Hz, 1 H, 4”-H), 6.78 (s, 1 H, 5-H), 6.65 (m, 2 H, 3”,6”-H), 6.50 (m, 2 H, 8-H, 5”-H), 6.35 (s, 2 H, 2’,6’-H), 5.96 (AB q, J = 1.2, 3.5 Hz, 2 H, OCHzO), 5.44 (5, 1 H, exchangeable, 4’-OH), 5.10 (s, 1 H, exchangeable, 2”-OH), 4.67 (d, J = 4.0 Hz, 1 H, 4-H), 4.61 (d, J = 4.8 Hz, 1 H, 1-H),4.38 (t, J = 8.5 Hz, 1 H, ll-H), 3.98 (t, J = 8.5 Hz, 1 H, 11-H), 3.79 (s,6 H, 3’,5’-OCH3), 3.24 (dd, J = 4.8, 14 Hz, 1 H, 2-H), 3.02 (m, 1 H, 3-H); MS m/z = 491 (M’). Anal. (C27H25N08) c , H. 4’-Demethyl-4843”-hydroxyanilino)-4-desoxypodophyllotoxin (12): yield 32%; amorphous powder from ether, mp 163-166 “C; IR (KBr) 3480 (OH), 3380 (NH), 2900 (aliphatic CH), 1750 (lactone), 1590, 1474 (aromatic C=C) cm-’; ‘H NMR (CDCl,) 6 7.05 (t, J = 8.0 Hz, 1 H, 5”-H), 6.78 (s, 1 H, 5-H), 6.52 (s, 1 H, 8-H), 6.33 (s,2 H, 2’,6’-H), 6.24 (dd, J = 2.2, 8.0 Hz, 1 H, 4”-H),
6.15(dd,J~1.7,8.0H~,1H,6”H),6.07(t,J~2.2H~,1H,2” 5.97 (d, J = 4.4 Hz, 2 H, OCHZO), 5.43 (s, 1 H, exchangeable, 4’-OH), 4.82 (s, 1 H, exchangeable, 3”-OH), 4.65 (d, J = 3.9 Hz, 1 H, 4-H), 4.58 (d, J = 4.8 Hz, 1 H, 1-H), 4.37 (t, J = 8.7 Hz, 1 H, 11-H),4.0 (t, J = 8.7 Hz, 1 H, 11-H), 3.79 (9, 6 H, 3’,5’-OCH,), 3.1 (dd, J = 4.8, 14.1 Hz, 1 H, 2-H), 2.98 (m, 1 H, 3-H); MS m/z = 491 (M+). Anal. (Cz7Hz5N08.Hz0) C, H.
4’-Demethyl-4~-(4”-hydroxyanilino)-4-desoxypodophyllotoxin (13): yield 33%; amorphous powder from ether, mp 162-165 “C; IR (KBr) 3525 (OH), 3345 (NH), 3010 (aromatic CH), 2900 (aliphatic CH), 1745 (lactone), 1600, 1475 (aromatic C-C) cm-’; ‘H NMR (DMSO-d6,DzO exchange) 6 6.69 (s, 1 H, 5-H), 6.55 (s, 4 H, 2”,3”,5”,6”-H), 6.48 (s, 1 H, 8-H), 6.23 (s, 2 H, 2’,6’-H), 5.94 (d, J = 9.7 Hz, 2 H, OCHZO), 4.68 (d, J = 4.3 Hz, 1 H, 4-H), 4.46 (d, J = 5.4 Hz, 1 H, 1-H), 4.29 (t, J = 7.6 Hz, 1 H, 11-H), 3.76 ( J = 7.6 Hz, 1 H, 11-H),3.61 (s,6 H, 3’,5’-OCH3), 3.28 (dd, J = 5.4, 15.8 Hz, 1H, 2-H), 2.95 (m, 1H, 3-H). Anal. (CnHwN08.Hz0) C, H. 4’-Demethyl-4842”-fluoroanilino)-4-desoxypodophyllotoxin (14): yield 43%; crystals from methanol, mp 197-198 “C; [aIz6D -128” (c = 0.25, CHCI,); 1R (KBr) 3500 (OH), 4480 (NH), 2890 (aliphatic C-H), 1755 (lactone), 1610, 1505, and 1475 (aromatic C - C ) cm-’; ‘H NMR (CDCl,) 6 7.04 (m, 2 H, 3”,6”-H),6.76 (s, 1 H, 5-H), 6.72 (m, 1 H, 5”-H), 6.60 (t, J = 7.2 Hz, 1 H, 4”-H), 6.54 (s, 1 H, 8-H), 6.34 (s, 2 H, 2’,6’-H), 5.97 (d, J = 7.3 Hz, 2 H, OCHzO),5.46 (s, 1 H, exchangeable, 4’-OH), 4.69 (d, J = 4.2 Hz, 1 H, 4-H), 4.62 (d, J = 4.9 Hz, 1 H, 1-H), 4.38 (t, J = 8.2 Hz, 1 H, 11-H), 4.10 (t, 1 H, exchangeable, NH), 3.82 (t, J = 8.2 Hz, 1 H, 11-H), 3.79 (s,6 H, 3’,5’-OCH,), 3.15 (dd, J = 5.0, 14.0 Hz, 1 H, 2-H), 3.00 (m, 1 H, 3-H). Anal. (CZ7Hz4FNO7) C, H, N. 4’-Demethyl-48-(3”-fluoroanilino)-4-desoxypodophyllotoxin (15): yield 60%; crystals from methanol, mp 201-203 “C dec; [.Iz5,, -132O (c = 1, CHCl,); IR (KB) 3500 (OH), 3360 (NH), 2900 (aliphatic C-H), 1750 (lactone), 1605, 1500, and 1475 (aromatic C=C) cm-’; ‘H NMR (CDCl,) 6 7.15 (t, J = 7.4 Hz, 1 H, 5”-H), 6.76 (s, 1H, 5-H), 6.53 (s, 1H, 8-H), 6.49 (dd, J = 1.2, 7.4 4”-H), J = ~ . O , ~ . O H Z , ~ H , O C H ~ O ) , ~ . ~ O ( ~ , J = Hz, ~ .1~H, HZ , ~ H6.40 , ~(s,-2HH,) 2’,6’-H), , ~ . ~ 6.32 ~ (d, J = 1.2 Hz, 1 H, 2”-H), 6.24 (dd, J = 1.2, 7.4 Hz, 1 H, 6”-H), 5.97 (AB q, J = 1.2, (d, J = 4.6 Hz, 1 H, 1-H), 4.38 (t, J = 6.0 Hz, 1 H, 11-H), 4.05 7.9 Hz, 2 H, OCHzO),5.44 (s, 1 H, exchangeable, 4’-OH), 4.67 (s, (t, J = 6.0 Hz, 1 H, 11-H),3.83 (s, 3 H, 4’-OCH3), 3.75 (s, 6 H, 1 H, exchangeable, NH), 4.63 (d, J = 4.0 Hz, 1 H, 4-H), 4.59 (d, 3’,5’-OCH,), 3.18 (dd, J = 4.6 Hz, 14.0 Hz, 1 H, 2-H), 3.0 (m, 1 J = 5.0 Hz, 1 H, 1-H), 4.39 (t, J = 8.5 Hz, 1 H, 11-H), 3.98 (t, J H, 3-H). Anal (Cz8Hz7N08.’/zHz0)C, H. = 8.5 Hz, 1 H, 11-H), 3.79 (s, 6 H, 3’,5’-OCH3),3.11 (dd, J = 5.0, 4’-Demethyl-4j3-bromo-4-desoxypodophyllotoxin (6). A 14.0 Hz, 1 H, 2-H), 3.00 (m, 1 H, 3-H). Anal. (C27H24FN07) C, solution of 4’-demethylepipodophylotoxin (10 g, 24 mmol) in 250 H, N. mL of dry dichloromethane was kept at 0 “C, and dry hydrogen 4’-Demethyl-4~-(4”-~uoroanilino)-4-desoxypodophyllobromide gas was bubbled through the solution. After 45 min, toxin (16): yield 45%; crystals from ethanol, mp 176-177 “C; nitrogen was also bubbled through the solution to drive off excess [a]25D -100” (c = 0.8, CHCI,); IR (KBr) 3540 (OH), 3420 (NH), hydrogen bromide. The solution was then evaporated in vacuo 2900 (aliphatic C-H), 1740 (latone), 1610,1500, and 1480 (aromatic to dryness, and the desired product (11.5 g) was obtained, which C=C) cm-’; ‘H NMR (CDC1,) 6 6.94 (t, J = 6.7, 2 H, 3”,5”-H), was used for the next reaction without further purification . 6.75 (s, 1 H, 5-H), 6.53 (s, 1 H, 8-H), 6.49 (q, J = 2.2, 6.2 Hz, 2 Synthesis of Compounds 11-23. A solution containing 4‘-
1368 Journal of Medicinal Chemistry, 1990, Vol. 33, No. 5 H, 2”,6”-H), 6.33 (9, 2 H, 2’,6’-H), 5.96 (AB q, J = 1.2, 7.5 Hz, 2 H, OCH20),5.43 (s, 1 H, exchangeable, 4’-OH), 4.60 (d, 2 H, 4-H and 1-H),4.37 (t, J = 7.5 Hz, 1 H, 11-H), 3.99 (t,J = 7.5 Hz, 1 H, 11-H), 3.79 (s, 6 H, 3’,5’-OCH3),3.73 (br, 1 H, exchangeable, NH), 3.13 (t,J = 5.0, 14.0 Hz, 1 H, 2-H), 3.00 (m, 1H, 3-H). Anal. (C27H24FN07) C, H, N. 4’-Demet hyl-48-(3”,5’~-difluoroanilino)-4-desoxypodophyllotoxin (17): yield 50%; crystals from ethanol, mp 180-183 “C; [cY]’~D-132” ( C = 0.33, CHCl,); IR (KBr) 3500 (OH), 3370 (NH), 2890 (aliphatic C-H), 1750 (lactone), 1620, 1590,1500,and 1475 (aromatic C=C) cm-’; ‘H NMR (CDCI,) 6.75 (s, 1 H, 5-H), 6.54 (s, 1 H, 8-H), 6.32 (s, 2 H, 2’,6’-H), 6.23 (m, 1 H, 4”-H), 6.07 (m, 2 H, 2”,6”-H), 5.98 (AB q, J = 1.3, 9.0 Hz, 2 H, OCH20),5.45 (s, 1 H, exchangeable, 4’-OH), 4.61 (m, 2 H, 4-H and 1-H), 4.39 (t,J = 8.5 Hz, 1 H, ll-H), 4.10 (d, J = 6.1 Hz, 1 H, exchangeable, NH), 3.85 (t, J = 8.5 Hz, 1 H, 11-H), 3.81 (s, 6 H, 3’,5’-OCH,), 3.08 (dd, J = 4.8, 14.1 Hz, 1 H, 2-H), 3.02 (m, 1 H, 3-H). Anal. (C27H23NF209) C, H, N. 4’-Demet hyl-4842”-chloroanilino)-4-desoxypodophyllotoxin (18): yield 38%; crystals from ethyl acetate-ether, mp 253-255 “C; [ a I z 5 D -90” (c = 1.0, CHCI,); IR (KBr) 3500 (OH), 3450 (NH), 2895 (aliphatic CH), 1751 (lactone), 1590,1500, and 1472 (aromatic C=C) cm-’; ‘H NMR (CDCI,) 7.31 (dd, J = 1.4, 7.9 Hz, 1 H, 3”-H), 7.18 (t,J = 8.8 Hz, 1 H, 5”-H), 6.76 (s, 1 H, 5-H), 6.73 (t, J = 9.0 Hz, 4”-H), 6.58 (d, J = 8.2 Hz, 1 H, 6”-H), 6.54 (s, 1 H, 8-H), 6.35 (s, 2 H, 2’,6’-H), 5.98 (AB q, J = 1.2, 4.2 Hz, 2 H, OCOH,O), 5.44 (s, 1 H, exchangeable, 4’-OH), 4.73 (t, J = 4.9 Hz, 1 H, 4-H), 4.64 (d, J = 4.9 Hz, 1 H, 1-H), 4.49 (d, J = 6.0 Hz, 1 H, exchangeable,NH), 4.36 (t,J = 8.3 Hz, 1 H, 11-H), 3.91 (t,J = 8.3 Hz, 1 H, 11-H), 3.80 (s, 6 H, 3’,5’-OCHJ, 3.17 (dd, J = 4.8, 14.0 Hz, 1 H, 2-H), 3.04 (m, 1 H, 3-H). Anal. (C27H24ClN07) C, H, N. 4’-Demethyl-4~-(3”-chloroanilino)-4-desoxypodophyllotoxin (19): yield 48%; crystals from ethyl acetate-ether, mp 174-176 “C; [fl]25D -112’ (c = 1.0, CHCI,); IR (KBr) 3500 (OH), 3360 (NH), 2920 (aliphatic CH), 1752 (lactone), 1580 and 1450 (aromatic C=C) cm-I; ‘H NMR (CDCl,) 7.12 (t, J = 8.1 Hz, 1 H, 5”-H), 6.76 (s, 1 H, 5-H), 6.74 (dd, J = 1.0,8.1 Hz, 1 H, 4”-H), 6.53 (br, 2 H, 8-H and 2”-H), 6.42 (dd, J = 1.6,6.5 Hz, 1 H, 6 - H ) , 6.33 (s, 2 H, 2’,6’-H), 5.97 (AB 9, J = 1.0, 8.7 Hz, 2 H, OCHZO), 5.43 (s, 1 H, exchangeable, 4’-OH), 4.66 (br, 1 H, 4-H), 4.59 (d, J = 4.8 Hz, 1 H, 1-H), 4.39 (t, J = 7.7 Hz, 1 H, 11-H), 3.99 (t,J = 7.7 Hz, 1 H, 11-H), 3.96 (br, 1 H, exchangeable, NH), 3.79 (s, 6 H, 3’,5’-OCH3),3.11 (dd, J = 5.8, 14.0 Hz, H, 2-H), 3.01 (m, 1 H, 3-H). Anal. (CZ7H2,C1NO7)C, H, N. 4’-Demethyl-48-( 4”-c hloroanilino)-4-desoxypodophyllotoxin (20): yield 52%; crystals from ethyl acetate-ether, mp 253-255 “C; [ c Y ] ~ -125” ~D (c = 0.75, CHClJ; IR (KB) 3500 (OH), 3360 (NH), 2900 (aliphatic CH), 1758 (lactone), 1605, 1590, and 1475 (aromatic C=C) cm-’; ‘H NMR (CDCl,) 6 7.17 (d, J = 8.7 Hz, 2 H, 3”,5”-H), 6.74 (s, 1 H, 5-H), 6.53 (s, 1 H, 8-H), 6.48 (d, J = 8.7 Hz, 2 H, 2”,6”-H), 6.32 (s, 2 H, 2’,6’-H), 5.96 (AB q, J = 1.0,6.8 Hz, 2 H, OCH20),5.43 (s, 1 H, exchangeable, 4’-OH),4.63 (d, J = 4.2 Hz, 1 H, 4-H), 4.59 (d, J = 4.9 Hz, 1 H, 1-H),4.38 (t, J = 8.0 Hz, 1 H, 11-H),3.96 ( J = 8.0 Hz, 1 H, 11-H), 3.79 (s, 6 H, 3’,5’-OCH3),3.12 (dd, J = 4.9, 14.1 Hz, 1 H, 2-H), 2.99 (m, 1 H, 3-H). Anal. (Cz7Hz4C1N07) C, H, N. 4’-Demethy1-48-(3”-bromoanilino)-4-desoxypodophyllotoxin (21): yield 55%; crystals from methanol-ether, mp 177-179
Lee et al.
“C; [ a I E D -105” (c = 1,CHCl,); IR (KBr) 3450 (OH), 3340 (NH), 2900 (aliphatic CH), 1740 (lactone), 1590,1500,and 1475 (aromatic C=C) cm-’; ‘H NMR (CDCl,) 6 7.07 (t,J = 8.0 Hz, 1 H, 5”-H), 6.90 (dd, J = 0.9, 7.9 Hz, 1 H, 4”-H), 6.75 (s, 1 H, 5-H), 6.70 (br, 1 H, 2”-H), 6.53 (s, 1H, 8-H), 6.47 (dd, J = 1.7,8.3Hz, 1H, 6”-H), 6.33 (s, 2 H, 2’,6’-H), 5.97 (dd, J = 1.2,9.3 Hz, 2 H, OCHzO), 5.43 (s, 1 H, exchangeable, 4’-OH), 4.65 (d, J = 4.2 Hz, 1 H, 4-H), 4.60 (d, J = 4.8 Hz, 1 H, 1-H), 4.39 (t, J = 7.3 Hz, 1 H, 11-H), 3.96 (t, J = 7.3 Hz, 1 H, 11-H), 3.90 (d, J = 6.2 Hz, 1 H, exchangeable, NH), 3.80 (s, 6 H, 3’,5’-OCH3), 3.10 (dd, J = 4.9, 14.0 Hz, 1 H, 2-H), 3.02 (m, 1 H, 3-H). Anal. (C2,HZ4BrNO7)C, H, N.
4‘-Demethyl-4~-(4”-bromoanilino)-4-desoxypodophyllotoxin (22): yield 57%; crystals from ethyl acetate-ethanol, mp 227-230 “C; [aIz5D -110’ (c = 0.5, CHCl,); IR (KBr) 3500 (OH), 3330 (NH), 2900 (aliphatic CH), 1755 (lactone), 1605,1590, and 1475 (aromatic C=C) cm-’; ‘H NMR (CDCI,) 6 7.30 (d, J = 8.9 Hz, 2 H, 3”,5”-H), 6.75 (s, 1,5-H), 6.53 (s, 1 H, 8-H), 6.44 (d, J = 8.9 Hz, 2 H, 2”,6”-H), 6.32 (s, 2 H, 2’,6’-H), 5.98 (AB q, J = 1.3,8.3 Hz, 2 H, OCH,O), 5.42 (s, 1 H, exchangeable,4’-OH), 4.62 (m, 2 H, 4-H and 1-H), 4.36 (t,J = 8.5 Hz, 1 H, 11-H), 3.95 (t, 8.5 Hz, 11-H), 3.86 (d, J = 7.8, 1H, exchangeable, NH), 3.79 (s, 6 H, 3’,5’-OCH,), 3.11 (dd, J = 4.8, 14.1 Hz, 1 H, 2-H), 3.00 (m, 1 H, 3-H). Anal. (CZ7H2,BrNO7)C, H, N. 4’-Demethyl-48-(4”-iodoanilino)-4-desoxypodophyllotoxin (23): yield 41%; crystals from ethanol, mp 198-200 “cdec; [ a I E D -111” (c = 0.5, CHCl,); IR (KBr) 3540 (OH), 3420 (NH), 2900 (aliphatic CH), 1770 (lactone), 1610, 1585, and 1480 (aromatic C=C) cm-’; ‘H NMR (CDClJ 6 7.46 ( d , J = 8.8 Hz, 2 H, 3’,5’-H), 6.74 (9, 1 H, 5-H), 6.65 (s, 1 H, 8-H), 6.35 (d, J = 8.8 Hz, 2 H, 2”,6”-H), 6.32 (s, 2 H, 2’,6’-H), 5.59 (AB q, J = 1.1,8.9 Hz, 2 H, OCH20),5.44 (s, 1 H, exchangeable, 4’-OH), 4.62 (m, 1 H, 4-H), 4.58 (d, J = 4.9 Hz, 1 H, 1-H), 4.34 (t, J = 8.5 Hz, 1 H, 11-HI, 3.94 (m, 2 H, 11-H, and NH), 3.78 (9, 6 H, 3’,5’-OCH3),3.09 (dd, J = 4.9,14.1 Hz, 1 H, 2-H), 2.99 (m, 1H, 3-H). Anal. (CnHNINO7) C, H, N. Biological Assay. Assays for the inhibition of human DNA topoisomerase I1 and the cellular protein-linked DNA breaks as well as the cytotoxicity in KB cells were carried out according to the procedures described previo~sly.~
Acknowledgment. The authors thank Mike Fisher of the Pharmacology Department, UNC-Chapel Hill, for KB cell culture assay. This work was supported by grants from the American Cancer Society (CH-370 B (K.-H.L.) and NIH NO. CA 44358 (Y.-C.C.)). Registry No. 2, 518-28-5; 6, 16477-16-0; 8, 125830-28-6;9, 125830-29-7;10, 125830-30-0; 11, 125830-31-1; 12, 125830-32-2; 13, 125830-33-3;14,125830-34-4; 15, 125830-35-5;16,125830-36-6; 17,125830-37-7;18,125830-38-8; 19,125830-39-9;20,125830-40-2; 21, 125830-41-3; 22, 125830-42-4;23, 125830-43-5; DNA topoisomerase, 80449-01-0; o-NH2C6H40H,95-55-6; m-NH2C6H40H, 591-27-5; P - N H ~ C ~ H ~ O 123-38-0; H, O - N H ~ C ~ H348-54-9; ~F, mNH&GH,F, 372-19-0; p-NH&H,F, 371-40-4; 3,5-F&H3NHz, 372-34-9; o - N H ~ C ~ H ~95-51-2; C ~ , m-NHZCsH4C1, 108-42-9; p NH2C6H4C1,106-47-8;m-NH2C6H4Br,591-19-5; p-NH2CsH4Br, 106-40-1;p-NH2C6H41,540-37-4; 4’-demethylepipodophyllotoxin, 6559-91-7.
